한수지 51(2), 135141, 18 Original Article Korean J Fish Aquat Sci 51(2),135141,18 쏙 (Upogebia major) 효소가수분해물의면역증강효과 이지현 양지은 송재희 1 맹상현 김소연 윤나영 국립수산과학원식품위생가공과, 1 서해수산연구소갯벌연구센터 Immunopotentiating Activities of Enzymatic Hydrolysate of Japanese Mud Shrimp Upogebia major JiHyun Lee, JiEun Yang, JaeHee Song 1, SangHyun Maeng, SoYeon Kim and NaYoung Yoon Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan 4683, Korea 1 Tidal Flat Research Center, National Institute of Fisheries Science, Gunsan 5414, Korea This study investigated the immunopotentiating activities of Japanese mud shrimp Upogebia major. We examined the effects of enzymatic hydrolysate from U. major on the production of nitric oxide (NO) and prostaglandin E 2 ( ) and on the expression of proinflammation cytokines including TNFα, IL6 and IL1β in RAW 264.7 cells. The treatment of six enzymatic hydrolysates of U. major (alcalase, αchymotrypsin [αchy], trypsin, pepsin, neutrase, protamex) significantly increased the production of NO in RAW 264.7 cells, with αchy having the greatest effect. This hydrolysate was fractionated by two ultrafiltration membranes at 3 and kda to created three fractions (below 3 kda, between 3 and kda, and above kda). Of these, the <3 kda and > kda fractions showed significant increases in NO production. These two fractions also induced production in RAW 264.7 cells and showed significant increases in the expression of all cytokines studied. These results suggest that enzymatic hydrolysate from U. major is a potentially useful food material with immunepotentiating effects. Key words: Upogebia major, Enzymatic hydrolysate, RAW 264.7 cells, Nitric Oxide, Cytokine 서론, (Delves and Roitt, ).,,, cytokine, T B (Parkin and Cohen, 1). cytokine (Plowden et al., 4). Nitric oxide (NO),,, T (Coleman, 1; Sharma et al., 7). NO,,,, cytokine, (Korhonen et al., 5). (Upogebia major),, (Hong, 13).,,, (Galasso et al., 17; Xia et al., 11). RAW 264.7 NO cytokine. https://doi.org/.5657/kfas.18.135 Korean J Fish Aquat Sci 51(2) 135141, April 18 This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial Licens (http://creativecommons.org/licenses/bync/3./) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 6 February 18; Revised 2 March 18; Accepted 13 March 18 Corresponding author: Tel: +82. 51. 7. 2652 Fax: +82. 51. 7. 2669 Email address: dbssud@korea.kr Copyright 18 The Korean Society of Fisheries and Aquatic Science 135 pissn:3748111, eissn:22878815
136 이지현ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ김소연ㆍ윤나영 재료및방법 재료 (U. major) 16 3., C. Alcalase 2.4L (Alc), chymotrypsin ( Chy), trypsin (Try), pepsin (Pep), serine, phthaldialdehyde (OPA), disodium tetraborate decahyarate, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), lipopolysaccharides (), griess reagent, cell proliferation reagent WST1 SigmaAldrich Chemical Co. (St. Louis, MO, USA), Neutrase (Neu), Protamex (Pro) Novo Co. (Novozyme Laboratories, Copenhagen, Denmark). RAW 264.7 (Korea Cell Line Bank, KCLB). fetal bovine serum (FBS), dulbecco s Modified Eagle s medium (DMEM) Gibco (Gibco BRL, USA). Penicillinstreptomycin solution Cellgro (Herndon, VA, USA)., TNF, IL6, IL1 kit R&D Systems Inc. (Minneapolis, MN, USA). 쏙의효소가수분해물제조 Kjeldahl, 41.39.69%. g 6 (Alc, Chy, Try, Pep, Neu, Pro), 1%. 6 Table 1 8,. 5, rpm.. 가수분해도 (Degree of hydrolysis, DH) AdlerNissen (1979). 7.62 g disodium tetraborate decahyarate mg SDS 15 ml 16 mg OPA 4 ml ethanol, 176 mg DTT ml OPA. 5 mg serine 5 ml serine (.9516 meqv/l). Serine 4 L OPA 3 ml 34 nm, OPA free amino groups (AdlerNissen, 1986). DH = h / h tot h tot protein equivalent peptide bonds, h. 가수분해물분획 ml 3 kda kda Ultrafiltration membranes (Amicon Ultrafilter devices; Millipore, Billerica, MA, USA) 3, rpm (<3 kda, 3 kda, > kda).. 총아미노산함량분석 6 N hydrochloric acid 1 24, 12..2 N sodium citrate buffer (ph 4.2).2 m membrane filter, (S43; Sykam, Eresing, Germany). 세포배양및세포독성 RAW 264.7 % heatinactivated FBS, 1% penicillin streptomycin DMEM 37, 5% CO 2. RAW 264.7 WST1 46 nm Microplate reader (BioTek, USA). Table 1. The conditions of enzymatic hydrolysis and degree of hydrolysis of the enzymatic hydrolysates of Japanese mud shrimp Upogebia major Enzyme Buffer ph Temperature ( C) DH (%) Alcalase.1M Na 2 8. 5 65.17±.84 Protamex.1M Na 2 8. 45 68.1±.43 Trypsin.1M Na 2 8. 37 64.56±.31 Neutrase.1M Na 2 8. 5 67.53±.28 Pepsin.1M GlycineHCl 2. 37 68.12±1.11 αchymotrypsin.1m Na 2 8. 37 69.81±.39 Enzymatic hydrolysates were obtained from 8 hour under the optical conditions. DH, degree of hydrolysis.
쏙효소가수분해물의면역증강효과 137 NO 생성효과 RAW 264.7 24 well plate 1 5 cells/well. 24 1 g/ml, 24. L Griess Microplate reader (BioTek, USA) 54 nm. sodium nitrite. 및 Cytokine 생성능 RAW 264.7 24 well plate 1 5 cells/well. 24 1 g/ml, 24., TNF, IL6, IL1 ELISA assay kit Microplate reader (BioTek, USA) 45 nm. 통계 (SD), Student ttest, P <.5 P <.1 Table 2. Total amino acids composition for the molecular weight cutoff fractions of the αchymotrypsin hydrolysates of Japanese mud shrimp Upogebia major (g/ g) Amino acids <3 kda 3 kda > kda Asp 6.93 4.72 7.83 Thr 3.65 1.94 3.86 Ser 2.37 2.19 3.8 Glu 6.67 4.88 8.7 Pro 2.57 1.89 3.67 Gly 1. 1.6 1.36 Ala 2.87 2.4 3.64 Cys.13.11.15 Val 2.93 2.37 3.9 Met.32.21.43 Ile 2.48 1.94 3.4 Leu 4.18 3.85 4.54 Tyr 2.91 1.38 1.82 Phe 3.24 3.3 3.28 His 3.19 3.11 3.46 Lys 3.25 2.93 3.63 Arg 3.87 2.63 2.9 Trp 2.84 2.54 3.58 Total 55.6 42.82 63.. 결과및고찰 효소가수분해및가수분해도 6 8, Table 1. Chy (69.81.39%)>Pep (68.12 1.11%)> Pro (68.1.43%)>Neu (67.53.28%) >Alc (65.17.84%)>Try (64.56.31%). 쏙효소가수분해물의세포독성및 NO 생성효과 nitric oxide (NO) 6 NO (Fig. 1). NO 6 RAW 264.7. 6 1 mg/ml RAW 264.7 9% ( ). 6 RAW 264.7 1 mg/ ml, (Blank).251 mg/ml,, Chy 25 15 5.25 mg/ml.5 mg/ml 1. mg/ml Blank Alc αchy Neu Pep Pro Try Fig. 1. Effects of the enzymatic hydrolysates of Japanese mud shrimp Upogebia major on nitric oxide production in RAW264.7 cells. All data are presented as means±sd of three independent experiments. P<.5 compared with nontreated group. P<.1, compared with treatment only., lipopolysaccharide; Alc, alcalase; αchy, αchymotrypsin; Neu, neutrase; Pep, pepsin; Pro, protamex; Try, trypsin.
138 이지현ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ김소연ㆍ윤나영 4 NO. 3 총아미노산함량 <3 kda (μg/ml) 12.5 25 5 Chy 3 kda kda membrane filter, (<3 kda, 3 kda, > kda ) (Table 2). Chy > kda (62.42 g/ g), <3 kda (55.6 g/ g), 3 kda (42.82 g/ g). 4 1 3 (ng/ml) 8 6 4 3 kda (μg/ml) 12.5 25 5 <3 kda (μg/ml) 12.5 25 5 (c) 4 1 3 (ng/ml) 8 6 4 > kda (μg/ml) 2.5 5 > kda (μg/ml) 2.5 5 Fig. 2. Effects of the molecular weight cutoff fractions of the αchymotrypsin hydrolysates of Japanese mud shrimp Upogebia major on nitric oxide production in RAW264.7 cells. Cells were treated with various concentration of <3 kda, 3 kda, and > kda (c) fractions of the αchymotrypsin hydrolysates of U. major. All data are presented as means±sd of three independent experiments. P<.5 compared with nontreated group. P<.1, compared with treatment only., lipopolysaccharide. Fig. 3. Effects of the molecular weight cutoff fractions of the αchymotrypsin hydrolysates of Japanese mud shrimp Upogebia major on prostaglandin E 2 ( ) release in RAW264.7 cells. Cells were treated with various concentration of <3 kda and > kda fractions of the αchymotrypsin hydrolysates of U. major. All data are presented as means±sd of three independent experiments. P<.5 compared with nontreated group. P<.1, compared with treatment only., lipopolysaccharide.
쏙효소가수분해물의면역증강효과 139 TNFα 5 5 4 4 TNFα (ng/ml) 3 TNFα (ng/ml) 3 <3 kda (μg/ml) 12.5 25 5 > kda (μg/ml) 2.5 5 IL6 8 8 6 6 IL6 (ng/ml) 4 IL6 (ng/ml) 4 <3k Da (μg/ml) 12.5 25 5 >k Da (μg/ml) 2.5 5 IL1β 5 5 4 4 IL1β (pg/ml) 3 IL1β (pg/ml) 3 <3k Da (μg/ml) 12.5 25 5 >k Da (μg/ml) 2.5 5 Fig. 4. Effects of cutoff fractions of Japanese mud shrimp the αchymotrypsin hydrolysates of Upogebia major on the production of proinflammatory cytokine (TNFα, IL6 and IL1β) in RAW264.7 cells. Cells were treated with various concentration of <3 kda and > kda fractions of the αchymotrypsin hydrolysates of U. major. All data are presented as means±sd of three independent experiments. P<.5 compared with nontreated group. P<.1, compared with treatment only. TNFα, tumor necrosis factor α; IL6, interleukin 6; IL1β, interleukin 1β;, lipopolysaccharide.
14 이지현ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ김소연ㆍ윤나영 peptide (Santiago Lopez et al., 16). Asp, Glu, Leu, Phe, His, Cys, Met. 쏙 αchy 가수분해물분획물의세포독성및 NO 생성효과 Chy 3 RAW 264.7, <3 kda 3 kda 5 g/ml 9%, > kda g/ml RAW 264.7 ( ). NO cytokine (Yu et al., 12). RAW 264.7 Chy 3, <3 kda (Blank) 12.55 g/ml NO, > kda 2.5 g/ml NO (Fig. 2). 3 kda NO. 생성효과 NO, cytokine (Harris et al., 2; Williams and Shacter, 1997). Chy 3 NO <3 kda > kda RAW 264.7 (Fig. 3). <3 kda (Blank) 12.55 g/ml, > kda 2.5 g/ml. <3 kda > kda RAW 264.7 NO. Cytokine 생성능 TNF, IL6, IL1 cytokine (Hanada and Yoshimura, 2). peptide,, cytokine (Cheung et al., 15; Wu et al., 15). Chy <3 kda > kda cytokine RAW 264.7 TNF, IL6, IL1, Fig. 4. Chy RAW 264.7 TNF <3 kda 12.55 g/ml TNF, > kda 2.5 g/ml TNF. RAW 264.7 Chy <3 kda > kda IL6 <3 kda 12.55 g/ml, > kda 2.5 g/ml. Chy <3 kda > kda RAW 264.7 IL1 <3 kda 12.55 g/ml., > kda 5 g/ml. Chy RAW 264.7 NO cytokine (TNF, IL6, IL1 ). 사사 (R186). References AdlerNissen J. 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27, 12561262. http://dx.doi. org/.21/jf6226a42. AdlerNissen J. 1986. Enzyme hydrolysis of food proteins. Elsevier Applied Sciences Publisher, New York, Barking, Essex, U.K., 1169. Cheung RCF, Ng TB and Wong JH. 15. Marine peptides: Bioactivities and applications. Mar Drugs 13, 46443. https://doi.org/.339/md13746 Coleman JW. 1. Nitric oxide in immunity and inflammation. Int Immunopharmacol 1, 1397146. https://doi. org/.16/s15675769(1)868.
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